Characterization of the human DHFR locus is nearly complete. A cosmid clone containing the 5 feet end of the gene was isolated allowing determination of DNA sequences of exon 1 and 2 and intron I as well as extensive 5 feet flanking sequence. The DNA sequence of the entire 2.9 kb 3 feet untranslated region has also been determined. By analysis of the cosmid clone, previously isolated Lamda clones, and restriction endonuclease mapping of genomic DNA, we have learned that the entire DHFR locus is 28 kb in length. The start site for transcription has been identified and the DHFR promoter sequences determined. The promoter is enhancer independent in a transient expression assay; a consensus core enhancer sequence has been identified within intron 1. Thorough characterization of the human DHFR gene has allowed its comparison to the mouse gene; homology in the coding sequences is striking but there is little if any homology in the 3 feet untranslated region and the introns. In contrast, homology between the human and mouse gene extends into the 5 feet flanking sequences for at least 350 base pairs. A third DHRF pseudogene has also been isolated by molecular cloning. DNA sequence determination has shown that this contains only exons 4, 5, and 6. A second insertion into the chromosomal site in which the intronless gene was inserted, resulted in interruption of the DHFR coding sequences by a member of the Alu moderately repetitive DNA sequence family.